Methods of treating vascular disease

ABSTRACT

The present invention relates to a method of treating patients suffering from, or at risk for, intimal hyperplasia and/or arteriosclerosis. The treatment includes administering a pharmaceutical composition that includes carbon monoxide to the patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/356,718 filed Feb. 13, 2002, which is incorporated herein byreference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NationalInstitutes of Health Grant Nos. HL55330, HL60234, HL67040, HL58688,HL53458, HL60234, HL5785405, and AI42365. The Government has certainrights in this invention.

TECHNICAL FIELD

This invention generally relates to treating vascular disease.

BACKGROUND

Heme oxygenase-1 (HO-1) catalyzes the first step in the degradation ofheme. HO-1 cleaves the α-meso carbon bridge of b-type heme molecules byoxidation to yield equimolar quantities of biliverdin IXa, carbonmonoxide (CO), and free iron. Subsequently, biliverdin is converted tobilirubin via biliverdin reductase, and the free iron is sequesteredinto ferritin (the production of which is induced by the free iron).

CO is recognized as an important signaling molecule (Verma et al.,Science 259:381-384, 1993). It has been suggested that carbon monoxideacts as a neuronal messenger molecule in the brain (Id.) and as aneuro-endocrine modulator in the hypothalamus (Pozzoli et al.,Endocrinology 735:2314-2317, 1994). Like nitric oxide, CO is a smoothmuscle relaxant (Utz et al., Biochem Pharmacol. 47:195-201, 1991;Christodoulides et al., Circulation 97:2306-9, 1995) and inhibitsplatelet aggregation (Mansouri et al., Thromb Haemost. 48:286-8, 1982).Inhalation of low levels of CO has been shown to have anti-inflammatoryeffects in some models.

Intimal hyperplasia, a thickening of the inner layer of the bloodvessel, is a pathological process that arises from vascular injurysubsequent to procedures such as angioplasty, bypass surgery or organtransplantation. Intimal hyperplasia continues to limit the success ofthese therapeutic interventions.

SUMMARY

The present invention is based, in part, on the discoveries that COprevents arteriosclerotic lesions and intimal hyperplasia followingaortic transplant and carotid artery balloon injury in animals.

Accordingly, in one aspect, the invention provides a method of treatingintimal hyperplasia in a patient. The method includes identifying apatient suffering from or at risk for intimal hyperplasia (e.g., intimalhyperplasia resulting from an angioplasty procedure or a transplantprocedure, or resulting from a procedure or condition other than atransplant procedure), and administering to the patient a pharmaceuticalcomposition comprising an amount of carbon monoxide effective to treatintimal hyperplasia in the patient.

The invention also provides a method of performing angioplasty in apatient. The method includes performing angioplasty in the patient, andbefore, during, and/or after performing angioplasty, administering tothe patient a pharmaceutical composition comprising an amount of COeffective to treat intimal hyperplasia in the patient. The angioplastycan be any angioplasty procedure, e.g., balloon angioplasty; laserangioplasty; artherectomy, e.g., directional atherectomy, rotationalatherectomy, or extraction atherectomy; and/or any angioplasty procedureusing a stent, or any combination of such procedures.

The invention also provides a method of treating (e.g., preventing ordecreasing) restenosis in a patient. The method includes providing avessel containing a pressurized gas comprising carbon monoxide gas,identifying a patient suffering from or at risk for restenosis,releasing the pressurized gas from the vessel to form an atmospherecomprising carbon monoxide gas, and exposing the patient to theatmosphere, wherein the amount of carbon monoxide in the atmosphere issufficient to treat restenosis in the patient.

In another aspect, the invention provides a method of treatingrestenosis in a patient. The method includes identifying a patientsuffering from or at risk for restenosis and administering to thepatient a pharmaceutical composition comprising an amount of carbonmonoxide effective to treat restenosis in the patient. Restenosis canresult from any angioplasty procedure, e.g., balloon angioplasty; laserangioplasty; artherectomy, e.g., directional atherectomy, rotationalatherectomy, or extraction atherectomy; and/or any angioplasty procedureusing a stent, or any combination of such procedures.

The invention also provides a method for performing vascular surgery,e.g., a transplant procedure, on a patient. The method includes: (a)performing vascular surgery (e.g., a transplant procedure) on a patient,and (b) before during and/or after (a), administering to the patient apharmaceutical composition comprising an amount of CO effective to treatarteriosclerosis (e.g., intimal hyperplasia) in the patient.

In another aspect, the invention provides a method of inhibiting smoothmuscle cell proliferation. The method includes providing a smooth musclecell(s), and administering to the smooth muscle cell(s) an amount of COeffective to inhibit proliferation of the smooth muscle muscle cell(s).This can be carried out in vivo or in vitro.

A method of performing angioplasty in a patient is also provided, whichincludes providing an angioplasty device (e.g, a device describedherein) capable of administering carbon monoxide to a patient,positioning the device in a blood vessel in need of angioplasty,performing angioplasty using the device, and before, during and/or afterperforming angioplasty, administering CO to the blood vessel using thedevice in an amount sufficient to treat intimal hyperplasia, to therebyperform angioplasty in the patient. The device can be any device capableof use in an angioplasty procedure, e.g., a device described herein.Alternatively or in addition, the device can be coated with aCO-releasing agent, e.g., a hydrogel, oil, or ointment, that releases COor a CO-releasing compound.

In another aspect, the invention provides a vessel comprising medicalgrade compressed CO gas. The vessel can bear a label indicating that thegas can be used to reduce restenosis, arteriosclerosis, and/or intimalhyperplasia in a patient (e.g., a human patient), and/or that it can beused in an angioplasty procedure. The CO gas can be in an admixture withnitrogen gas, with nitric oxide and nitrogen gas, or with anoxygen-containing gas. The CO gas can be present in the admixture at aconcentration of at least about 0.025%, e.g., at least about 0.05%,0.10%, 0.50%, 1.0%, 2.0%, 10%, 50%, or 90%.

The invention also provides a kit that includes an angioplasty device(e.g., a balloon angioplasty device; a laser angioplasty device; anatherectomy device; and/or a stent) and a vessel containing CO (e.g., aliquid and/or gaseous CO composition). The angioplasty device is capableof administering carbon monoxide to a patient. The kit can furtherinclude instructions for use of the carbon monoxide composition in amethod for performing angioplasty in a patient.

In another aspect, the invention provides angioplasty devices (e.g.,balloon angioplasty devices, laser angioplasty devices, atherectomydevices, and stents, e.g., a device described herein) capable ofadministering CO to a patient and/or a blood vessel immediately before,during, and/or after an angioplasty procedure. In one embodiment, theangioplasty device comprises a CO composition. In another embodiment,the device is a balloon angioplasty device that includes an inflatablemember (e.g., a balloon) having a plurality of apertures, and areservoir containing CO (e.g., a liquid or gaseous CO composition)connected to the inflatable member, such that CO can be delivered fromthe reservoir through the inflatable member and to the blood vessel.

Also within the invention is the use of CO in the manufacture of amedicament for treatment or prevention of a condition described herein,e.g., intimal hyperplasia, restenosis, and/or arteriosclerosis. Themedicament can also be used in a method for performing an angioplastyprocedure and/or a transplantation procedure. The medicament can be inany form as described herein, e.g., a liquid or gaseous CO composition.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Suitable methods and materialsare described below, although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photomicrograph (50× magnification) of a syngeneicallytransplanted aortic graft illustrating the effect of syngeneictransplantation on the graft.

FIG. 1B is a photomicrograph (50× magnification) of an allogeneicallytransplanted aortic graft illustrating the effect of allogeneictransplantation on the graft.

FIG. 1C is a photomicrograph (50× magnification) of an allogeneicallytransplanted aortic graft illustrating the effect of allogeneictransplantation on the graft when the recipient is exposed to CO.

FIG. 1D is a photomicrograph (200× magnification) of a syngeneicallytransplanted aortic graft illustrating the effect of syngeneictransplantation on the graft.

FIG. 1E is a photomicrograph (200× magnification) of an allogeneicallytransplanted aortic graft illustrating the effect of allogeneictransplantation on the graft.

FIG. 1F is a photomicrograph (200× magnification) of an allogeneicallytransplanted aortic graft illustrating the effect of allogeneictransplantation on the graft when the recipient is exposed to CO.

FIG. 1G is a bar graph illustrating the mean relative areas (inarbitrary units) of the intima of aortic grafts transplantedsyngeneically into air-exposed recipients (Syng.), allogeneically intoair-exposed recipients (Allo.), and allogeneically into CO-exposedrecipients (Allo.+CO).

FIG. 1H is a bar graph illustrating the mean relative areas (inarbitrary units) of the media of aortic grafts transplantedsyngeneically into air-exposed recipients (Syng.), allogeneically intoair-exposed recipients (Allo.), and allogeneically into CO-exposedrecipients (Allo.+CO).

FIG. 1I is a bar graph illustrating the intima/media area ratio ofaortic grafts transplanted syngeneically into air-exposed recipients(Syng.), allogeneically into air-exposed recipients (Allo.), andallogeneically into CO-exposed recipients (Allo.+CO).

FIG. 2A is a bar graph illustrating accumulation of activated leukocytes(measured by counting total nuclei) in the adventitia of aortic graftstransplanted syngeneically into air-exposed recipients (Syngeneic),allogeneically into air-exposed recipients (Allogeneic) andallogeneically into recipients exposed to various concentrations ofCO(CO 250 ppm; CO 500 ppm; and CO 750-1000 ppm).

FIG. 2B is a set of bar graphs illustrating accumulation of CD45, ED1,MHCII, and CD54 positive cells in the adventitia of aortic graftstransplanted syngeneically into air-exposed recipients (Syng.),allogeneically into air-exposed recipients (Allog.), and allogeneicallyinto CO-exposed recipients (Allo.+CO).

FIG. 2C is a set of bar graphs illustrating accumulation of CD3, CD4,and CD8 positive cells in the adventitia of aortic grafts transplantedsyngeneically into air-exposed recipients (Syng.), allogeneically intoair-exposed recipients (Allog.), and allogeneically into CO-exposedrecipients (Allo.+CO).

FIG. 3A is a photomicrograph (10× magnification) of a carotid arterysample illustrating the effect of balloon angioplasty on the artery.

FIG. 3B is a photomicrograph (10× magnification) of a carotid arterysample illustrating the effect of balloon angioplasty on the artery whenthe subject is pre-exposed to CO.

FIG. 3C is a photomicrograph of a carotid artery sample illustrating theeffect of balloon angioplasty on the artery.

FIG. 3D is a photomicrograph of a carotid artery sample illustrating theeffect of balloon angioplasty on the artery when the subject ispre-exposed to CO.

FIG. 3E is a bar graph illustrating the mean relative areas (inarbitrary units) of the intima of carotid arteries subjected to balloonangioplasty when the subject animal is pre-exposed either to air(Control) or 250 ppm CO(CO).

FIG. 3F is a bar graph illustrating the mean relative areas (inarbitrary units) of the media of carotid arteries subjected to balloonangioplasty when the subject animal is pre-exposed either to air(Control) or 250 ppm CO(CO).

FIG. 3G is a bar graph illustrating the intima/media area ratio ofcarotid arteries subjected to balloon angioplasty when the subjectanimal is pre-exposed either to air (Control) or 250 ppm CO(CO).

FIG. 4A is a line graph illustrating proliferation of rat SMC that werenon-transduced (o; Medium), transduced with Lac.Z recombinant adenovirus(□; LacZ Rec. Ad.) or transduced with HO-1 recombinant adenovirus (;HO-1 rec. Ad.).

FIG. 4B is a line graph illustrating proliferation of rat SMC in thepresence (; 1000 ppm) or absence (□) of CO.

FIG. 4C is a bar graph illustrating proliferation of SMC isolated fromwild type (WT) or HO-1 deficient (ho-1^(−I−)) mice in the presence (CO)and absence (Air) of CO.

FIG. 4D is a Western blot illustrating the effect of CO exposure (for 0,4, 5, 16, and 24 hours) on p21 and β-actin protein expression in mouseSMC.

FIG. 4E is a bar graph illustrating the effect of CO on proliferation ofmouse SMC isolated from wild type (wt), p21Cip1 (p21^(−/−)) and p53(p53^(−/−)) deficient mice. Gray bars indicate cells exposed to room airand black bars cells exposed to CO (250 ppm).

FIG. 4F is a bar graph illustrating the intima/media area ratio ofballoon-injured carotid arteries from wild type (C57/B16/S129; wt) andp21^(−/−) mice exposed to air (Air) or carbon monoxide (CO).

FIG. 5A is a bar graph illustrating the effect of air (Air) and CO (250ppm for 8 h or 16 h) exposure on the mean cellular cGMP content of mouseSMCs.

FIG. 5B is a bar graph illustrating [³H]thymidine uptake by mouse SMCsexposed to air (Air), CO (250 ppm) and CO plus the guanylate cyclaseinhibitor 1H(1,2,4) Oxadiazolo(4,3-a) Quinoxalin-1 (CO/ODQ).

FIG. 5C is a composite picture of a Western blot illustrating the effectof 8-Bromoguanosine 3′-5′-cyclic monophosphate sodium salt (8-Br-cGMP)on p21^(Cip1) expression.

FIG. 5D is a bar graph illustrating [³H]thymidine uptake by SMC isolatedfrom wild type (wt) and p21^(Cip1) (p21^(−/−)) deficient mice in thepresence (8Br-cGMP; 8Br-cGMP (p21^(−/−))) and absence (Air) of the cGMPanalogue 8Br-cGMP.

FIG. 5E is a composite picture of a Western blot illustrating the effectof CO (250 ppm) on expression of phosphorylated p38 MAPK (p-p38), ATF-2(p-ATF-2), JNK (p-JNK) and ERK (p-ERK) as compared to total p38 MAPK,ATF-2, JNK, and ERK in SMC.

FIG. 5F is a bar graph illustrating [³H]thymidine uptake by mouse SMCexposed air (Air) 250 ppm CO(CO) and CO plus the p38 MAPK inhibitorSB203580 (CO/SB).

FIG. 5G is a composite picture of a Western blot illustrating the effectof air (Air), CO (250 ppm; CO)), SB203580, and DMSO on expression ofp21^(Cip1) in mouse SMC.

FIG. 6A is a composite picture of a Western blot illustrating the effectof 8-Br-cGMP on expression of p38 MAPK in mouse SMC.

FIG. 6B is a bar graph illustrating [³H]thymidine uptake by mouse SMCexposed to air (Air), CO plus 8-Br-cGMP (8-Br-cGMP), and CO plus8-Br-cGMP plus SB203580 (8-Br-cGMP+SB203580).

FIG. 7A is a flow cytometry plot illustrating the effect of air (Air)and CO (250 ppm) exposure on the cell cycle of rat aortic SMC.

FIG. 7B is a line graph illustrating the effect of air (□) and CO (▪) onSMC proliferation.

FIG. 8 is a bar graph illustrating [³H]thymidine uptake by wild type(wt), p21^(−/−), and p53^(−/−) mouse SMC exposed to either air (graybars) or to CO (250 ppm; black bars) for 24 hours.

FIG. 9A is a photomicrograph (20× magnification) of a carotid arterysection from a wild type mouse 14 days after wire injury. The subjectanimal was exposed to room air for 1 h prior to wire injury.

FIG. 9B is a photomicrograph (20× magnification) of a carotid arterysection from a wild type mouse 14 days after wire injury. The subjectanimal was exposed to CO (250 ppm) for 1 h prior to wire injury.

FIG. 9C is a photomicrograph (20× magnification) of a carotid arterysection from a p21^(−/−) mouse 14 days after wire injury. The subjectanimal was exposed to room air for 1 h prior to wire injury.

FIG. 9D is a photomicrograph (20× magnification) of a carotid arterysection from a p21^(−/−) mouse 14 days after being subjected to wireinjury. The subject animal was exposed to CO (250 ppm) for 1 h prior towire injury.

FIG. 9E is a bar graph illustrating the mean relative areas (inarbitrary units) of the intima of wire-injured carotid arteries fromwild type (wt) and p21^(−/−) mice exposed to either air (Control) or 250ppm CO(CO).

FIG. 9F is a bar graph illustrating the mean relative areas (inarbitrary units) of the media of wire-injured carotid arteries from wildtype (wt) and p21^(−/−) mice exposed to either air (Control) or 250 ppmCO(CO).

FIG. 9G is a bar graph illustrating the intima/media area ratio ofwire-injured carotid arteries from wild type (wt) and p21^(−/−) miceexposed to either air (Control) or 250 ppm CO(CO).

FIG. 10 is a bar graph illustrating [³H]thymidine uptake by p21^(−/−)mouse-derived SMC treated with air (Air), CO (250 ppm; CO) and CO plusSB203580 (CO/SB).

FIG. 11A is a bar graph illustrating [³H]thymidine uptake by wild type(wt), enos^(−/−), and inos^(−/−) deficient mouse-derived SMC exposed toair (Air) or CO (250 ppm; CO).

FIG. 11B is a bar graph illustrating the mean intimal and media areas,and the intima/media area ratios, of carotid arteries fromSprague-Dawley rats exposed to room air (white bars) or NO (Black bars;1 hour; 250 ppm) prior to balloon injury of the carotid artery.

FIG. 12A is a composite picture of a Western blot illustrating PAI-1expression in SMC treated with and without serum (Serum and No Serum,respectively) and with and without CO (Air and CO, respectively) for 24or 48 hours. Liver=whole cell lysates from rat liver homogenates treatedwithout endotoxin (LPS). Liver+LPS=whole cell lysates from rat liverhomogenates treated with endotoxin (LPS). TNF-α=control wherein TNF-αwas added to the cell culture to stimulate expression of PAI-1.

FIG. 12B is a picture of a Western blot illustrating PAI-1 expression inuntransplanted (control), transplanted (Allo.+Air), and CO-treatedtransplanted (Allo.+CO) aortas 56 days after transplantation.

FIG. 12C is a picture of a Commassie blue stained polyacrylamide gelused for the Western blot of FIG. 12B, which illustrates PAI-expressionin untransplanted (control), transplanted (Allo.+Air), and CO-treatedtransplanted (Allo.+CO) aortas 56 days after transplantation.

FIGS. 13A-13B illustrate an example of balloon angioplasty devicecapable of administering CO to a patient during an angioplastyprocedure, at various stages of operation.

FIGS. 13C-13D illustrate alternative embodiments of the balloonangioplasty device.

FIGS. 14A-14B illustrate an example of a stent capable of administeringCO to a patient during an angioplasty procedure, at various stages ofoperation.

FIG. 15 illustrates an example of balloon angioplasty device withmultiple balloons designed to administer CO to a patient during anangioplasty procedure.

FIG. 16 illustrates an example of an atherectomy device capable ofadministering CO to a patient during an angioplasty procedure.

DETAILED DESCRIPTION

The term “carbon monoxide” (or “CO”) as used herein describes molecularcarbon monoxide in its gaseous state, compressed into liquid form, ordissolved in aqueous solution. The term “carbon monoxide composition” or“pharmaceutical composition comprising carbon monoxide” is usedthroughout the specification to describe a gaseous or liquid compositioncontaining carbon monoxide that can be administered to a patient and/ora blood vessel, e.g, a patient (or blood vessel) subjected toangioplasty, bypass surgery, transplant, or any other procedure thatmay/will result in intimal hyperplasia and/or arteriosclerosis. Askilled practitioner will recognize which form of the pharmaceuticalcomposition, e.g., gaseous, liquid, or both gaseous and liquid forms, ispreferred for a given application.

The term “intimal hyperplasia” is an art-recognized term and is usedherein to refer to proliferation of cells, e.g., smooth muscle cells,within the intima of a blood vessel. The skilled practitioner willappreciate that intimal hyperplasia can be caused by any number offactors, e.g., mechanical, chemical and/or immunological damage to theintima. Intimal hyperplasia can often be observed in patients, forexample, following balloon angioplasty or vascular surgery, e.g.,vascular surgery involving vein grafts (e.g., transplant surgery).“Arteriosclerosis,” “arteriosclerotic lesion,” “arterioscleroticplaque,” and “arteriosclerotic condition” are also art recognized termterms, and are used herein to describe a thickening and hardening of thearterial wall. The term “vasculature” as used herein refers to thevascular system (or any part thereof) of a body, human or non-human, andincludes blood vessels, e.g., arteries, arterioles, veins, venules, andcapillaries. The term “restenosis” refers to re-narrowing of an arteryfollowing angioplasty.

The term “angioplasty” is an art-recognized term and refers to anyprocedure, singly or in combination, involving remodeling of a bloodvessel, e.g., dilating a stenotic region in a patient's vasculature torestore adequate blood flow beyond the stenosis. Such procedures includepercutaneous transluminal angioplasty (PTA), which employs a catheterhaving an expansible distal end, i.e., an inflatable balloon (known as“balloon angioplasty”); laser angioplasty; extraction atherectomy;directional atherectomy; rotational atherectomy; stenting; and any otherprocedure for remodeling a blood vessel, e.g., an artery.

The terms “effective amount” and “effective to treat,” as used herein,refer to an amount or a concentration of carbon monoxide utilized for aperiod of time (including acute or chronic administration and periodicor continuous administration) that is effective within the context ofits administration for causing an intended effect or physiologicaloutcome. Effective amounts of carbon monoxide for use in the presentinvention include, for example, amounts that prevent or reduce intimalhyperplasia following a procedure, e.g., angioplasty. Effective amountsof carbon monoxide also include amounts that prevent or reducearteriosclerosis in a patient, e.g., a transplant patient. The term“treat(ment)” is used herein to describe delaying the onset of,inhibiting, or alleviating the detrimental effects of a condition, e.g.,intimal hyperplasia and/or arteriosclerosis.

For gases, effective amounts of CO generally fall within the range ofabout 0.0000001% to about 0.3% by weight, e.g., 0.0001% to about 0.25%by weight, preferably at least about 0.001%, e.g., at least about0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.10%,0.15%, 0.20%, 0.22%, or 0.24% by weight of CO. Preferred ranges of COinclude 0.002% to about 0.24%, about 0.005% to about 0.22%, about 0.01%to about 0.20%, and about 0.02% to about 0.1% by weight. For liquidsolutions of CO, effective amounts generally fall within the range ofabout 0.0001 to about 0.0044 g CO/100 g liquid, e.g., at least about0.0001, 0.0002, 0.0004, 0.0006, 0.0008, 0.0010, 0.0013, 0.0014, 0.0015,0.0016, 0.0018, 0.0020, 0.0021, 0.0022, 0.0024, 0.0026, 0.0028, 0.0030,0.0032, 0.0035, 0.0037, 0.0040, or 0.0042 g CO/100 g aqueous solution.Preferred ranges include, e.g., about 0.0010 to about 0.0030 g CO/100 gliquid, about 0.0015 to about 0.0026 g CO/100 g liquid, or about 0.0018to about 0.0024 g CO/100 g liquid. A skilled practitioner willappreciate that amounts outside of these ranges may be used dependingupon the application.

The term “patient” is used throughout the specification to describe ananimal, human or non-human, to whom treatment according to the methodsof the present invention is provided. Veterinary and non-veterinaryapplications are contemplated by the present invention. The termincludes but is not limited to mammals, e.g., humans, other primates,pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters,cows, horses, cats, dogs, sheep and goats.

The term “transplantation” is used throughout the specification as ageneral term to describe the process of transferring an organ or tissueinto a patient. The term “transplantation” is defined in the art as thetransfer of living tissues or cells from a donor to a recipient, withthe intention of maintaining the functional integrity of thetransplanted tissue or cells in the recipient (see, e.g., The MerckManual, Berkow, Fletcher, and Beers, Eds., Merck Research Laboratories,Rahway, N.J., 1992). The term includes all categories of transplantsknown in the art. Transplants are categorized by site and geneticrelationship between donor and recipient. The term includes, e.g.,autotransplantation (removal and transfer of cells or tissue from onelocation on a patient to the same or another location on the samepatient), allotransplantation (transplantation between members of thesame species), and xenotransplantation (transplantations between membersof different species).

The term “donor” or “donor patient” as used herein refers to an animal(human or non-human) from whom an organ or tissue can be obtained forthe purposes of transplantation to a recipient patient. The term“recipient” or “recipient patient” refers to an animal (human ornon-human) into which an organ or tissue can be transferred.

The terms “organ rejection”, “transplant rejection” and “rejection” areart-recognized and are used throughout the specification as generalterms to describe the process of rejection of an organ, tissues, orcells in a recipient. Included within the definition are, for example,three main patterns of rejection that are typically identified inclinical practice: hyperacute rejection, acute rejection, and chronicrejection (see, e.g., Oxford Textbook of Surgery, Morris and Malt, Eds.,Oxford University Press (1994)).

The term “organ(s)” is used throughout the specification as a generalterm to describe any anatomical part or member having a specificfunction in the animal. Further included within the meaning of this termare substantial portions of organs, e.g., cohesive tissues obtained froman organ. Such organs include but are not limited to kidney, liver,heart, intestine, e.g., large or small intestine, pancreas, and lungs.Also included in this definition is vasculature, e.g., veins andarteries, and bones.

Individuals considered at risk for developing intimal hyperplasia orarteriosclerosis may benefit particularly from the invention, primarilybecause prophylactic CO treatment can be administered before a procedureis performed on a patient or before there is any evidence of intimalhyperplasia or an arteriosclerotic plaque. Individuals “at risk”include, e.g., patients that have or will have any type of mechanical,chemical and/or immunological damage to the intima, e.g., patients thatwill or have undergone transplant surgery and/or angioplasty. Skilledpractitioners will appreciate that a patient can be determined to be atrisk for intimal hyperplasia or arteriosclerosis by any method known inthe art, e.g., by a physician's diagnosis.

Preparation of Gaseous Compositions

A CO composition may be a gaseous composition. Compressed or pressurizedgas useful in the methods of the invention can be obtained from anycommercial source, and in any type of vessel appropriate for storingcompressed gas. For example, compressed or pressurized gases can beobtained from any source that supplies compressed gases, such as oxygen,for medical use. The term “medical grade” gas, as used herein, refers togas suitable for administration to patients as defined herein. Thepressurized gas including CO used in the methods of the presentinvention can be provided such that all gases of the desired finalcomposition (e.g., CO, He, NO, CO₂, O₂, N₂) are in the same vessel,except that NO and O₂ cannot be stored together. Optionally, the methodsof the present invention can be performed using multiple vesselscontaining individual gases. For example, a single vessel can beprovided that contains carbon monoxide, with or without other gases, thecontents of which can be optionally mixed with the contents of othervessels, e.g., vessels containing oxygen, nitrogen, carbon dioxide,compressed air, or any other suitable gas or mixtures thereof.

Gaseous compositions administered to a patient according to the presentinvention typically contain 0% to about 79% by weight nitrogen, about21% to about 100% by weight oxygen and about 0.0000001% to about 0.3% byweight (corresponding to about 1 ppb or 0.001 ppm to about 3,000 ppm)CO. Preferably, the amount of nitrogen in the gaseous composition isabout 79% by weight, the amount of oxygen is about 21% by weight and theamount of CO is about 0.0001% to about 0.25% by weight. The amount of COis preferably at least about 0.001%, e.g., at least about 0.005%, 0.01%,0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.10%, 0.15%, 0.20%,0.22%, or 0.24% by weight. Preferred ranges of CO include 0.005% toabout 0.24%, about 0.01% to about 0.22%, about 0.015% to about 0.20%,and about 0.025% to about 0.1% by weight. It is noted that gaseous COcompositions having concentrations of CO greater than 0.3% (such as 1%or greater) may be used for short periods (e.g., one or a few breaths),depending upon the application.

A gaseous CO composition may be used to create an atmosphere thatcomprises CO gas. An atmosphere that includes appropriate levels of COgas can be created, for example, by providing a vessel containing apressurized gas comprising CO gas, and releasing the pressurized gasfrom the vessel into a chamber or space to form an atmosphere thatincludes the CO gas inside the chamber or space. Alternatively, thegases can be released into an apparatus that culminates in a breathingmask or breathing tube, thereby creating an atmosphere comprising CO gasin the breathing mask or breathing tube, ensuring the patient is theonly person in the room exposed to significant levels of CO.

CO levels in an atmosphere can be measured or monitored using any methodknown in the art. Such methods include electrochemical detection, gaschromatography, radioisotope counting, infrared absorption, colorimetry,and electrochemical methods based on selective membranes (see, e.g.,Sunderman et al., Clin. Chem. 28:2026-2032, 1982; Ingi et al., Neuron16:835-842, 1996). Sub-parts per million CO levels can be detected by,e.g., gas chromatography and radioisotope counting. Further, it is knownin the art that CO levels in the sub-ppm range can be measured inbiological tissue by a midinfrared gas sensor (see, e.g., Morimoto etal., Am. J. Physiol. Heart. Circ. Physiol 280:H482-H488, 2001). COsensors and gas detection devices are widely available from manycommercial sources.

Preparation of Liquid Compositions

A pharmaceutical composition comprising CO may also be a liquidcomposition. A liquid can be made into a pharmaceutical compositioncomprising CO by any method known in the art for causing gases to becomedissolved in liquids. For example, the liquid can be placed in aso-called “CO₂ incubator” and exposed to a continuous flow of CO,preferably balanced with carbon dioxide, until a desired concentrationof CO is reached in the liquid. As another example, CO gas can be“bubbled” directly into the liquid until the desired concentration of COin the liquid is reached. The amount of CO that can be dissolved in agiven aqueous solution increases with decreasing temperature. As stillanother example, an appropriate liquid may be passed through tubing thatallows gas diffusion, where the tubing runs through an atmospherecomprising CO (e.g., utilizing a device such as an extracorporealmembrane oxygenator). The CO diffuses into the liquid to create a liquidCO composition.

It is likely that such a liquid composition intended to be introducedinto a living animal will be at or about 37° C. at the time it isintroduced into the animal.

The liquid can be any liquid known to those of skill in the art to besuitable for administration to patients (see, for example, OxfordTextbook of Surgery, Morris and Malt, Eds., Oxford University Press(1994)). In general, the liquid will be an aqueous solution. Examples ofsolutions include Phosphate Buffered Saline (PBS), Celsior™, Perfadex™,Collins solution, citrate solution, and University of Wisconsin (UW)solution (Oxford Textbook of Surgery, Morris and Malt, Eds., OxfordUniversity Press (1994)). In one embodiment of the present invention,the liquid is Ringer's Solution, e.g., lactated Ringer's Solution, orany other liquid that can be used infused into a patient. In anotherembodiment, the liquid includes blood, e.g., whole blood.

Any suitable liquid can be saturated to a set concentration of CO viagas diffusers. Alternatively, pre-made solutions that have been qualitycontrolled to contain set levels of CO can be used. Accurate control ofdose can be achieved via measurements with a gas permeable, liquidimpermeable membrane connected to a CO analyzer. Solutions can besaturated to desired effective concentrations and maintained at theselevels.

Treatment of Patients and Vasculature with CO Compositions

The present invention contemplates administering CO compositions topatients and/or portions of their vasculature before, during, and/orafter the patient undergoes angioplasty, transplant surgery, vascularsurgery, or any other procedure that causes/increases the risk ofintimal hyperplasia, restenosis, and/or arteriosclerosis in the patient.A patient can be treated systemically with gaseous and/or liquid COcompositions by any method known in the art for administering gasesand/or liquids to patients, e.g., by inhalation of the gas andintravenous or intraarterial administration of the liquid. With systemictreatment, substantially all of the patient's vasculature can be treatedwith CO. A portion of a patient's vasculature, e.g., a specific vein orartery, can be treated by administering a gaseous or liquid COcomposition directly to the vein or artery. Although the presentinvention is not limited to any particular mode for administering COcompositions to patients and/or portions of their vasculature, varioustreatments are discussed in detail below.

Systemic Delivery of Gaseous CO

Gaseous CO compositions can be delivered systemically to a patient,e.g., a patient suffering from or at risk for intimal hyperplasia (e.g.,restenosis and/or arteriosclerosis). Gaseous CO compositions aretypically administered by inhalation through the mouth or nasal passagesto the lungs, where the CO is readily absorbed into the patient'sbloodstream. The concentration of active compound (CO) utilized in thetherapeutic gaseous composition will depend on absorption, distribution,inactivation, and excretion (generally, through respiration) rates ofthe CO as well as other factors known to those of skill in the art. Itis to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.Acute, sub-acute and chronic administration of CO are contemplated bythe present invention. CO can be delivered to the patient for a time(including indefinitely) sufficient to treat the condition and exert theintended pharmacological or biological effect.

The following are examples of some methods and devices that can beutilized to administer gaseous CO compositions to patients.

Ventilators

Medical grade CO (concentrations can vary) can be purchased mixed withair or another oxygen-containing gas in a standard tank of compressedgas (e.g., 21% O₂, 79% N₂). It is non-reactive, and the concentrationsthat are required for the methods of the present invention are wellbelow the combustible range (10% in air). In a hospital setting, the gaspresumably will be delivered to the bedside where it will be mixed withoxygen or house air in a blender to a desired concentration in ppm(parts per million). The patient will inhale the gas mixture through aventilator, which will be set to a flow rate based on patient comfortand needs. This is determined by pulmonary graphics (i.e., respiratoryrate, tidal volumes etc.). Fail-safe mechanism(s) to prevent the patientfrom unnecessarily receiving greater than desired amounts of carbonmonoxide can be designed into the delivery system. The patient's COlevel can be monitored by studying (1) carboxyhemoglobin (COHb), whichcan be measured in venous blood, and (2) exhaled CO collected from aside port of the ventilator. CO exposure can be adjusted based upon thepatient's health status and on the basis of the markers. If necessary,CO can be washed out of the patient by switching to 100% O₂ inhalation.CO is not metabolized; thus, whatever is inhaled will ultimately beexhaled except for a very small percentage that is converted to CO₂. COcan also be mixed with any level of O₂ to provide therapeutic deliveryof CO without consequential hypoxic conditions.

Face Mask and Tent

A CO-containing gas mixture is prepared as above to allow passiveinhalation by the patient using a facemask or tent. The concentrationinhaled can be changed and can be washed out by simply switching over to100% O₂. Monitoring of CO levels would occur at or near the mask or tentwith a fail-safe mechanism that would prevent too high of aconcentration of CO from being inhaled.

Portable Inhaler

Compressed CO can be packaged into a portable inhaler device and inhaledin a metered dose, for example, to permit intermittent treatment of arecipient who is not in a hospital setting. Different concentrations ofCO could be packaged in the containers. The device could be as simple asa small tank (e.g., under 5 kg) of appropriately diluted CO with anon-off valve and a tube from which the patient takes a whiff of COaccording to a standard regimen or as needed.

Intravenous Artificial Lung

An artificial lung (a catheter device for gas exchange in the blood)designed for O₂ delivery and CO₂ removal can be used for CO delivery.The catheter, when implanted, resides in one of the large veins andwould be able to deliver CO at desired concentrations either forsystemic delivery or at a local site. The delivery can be a localdelivery of a high concentration of CO for a short period of time at thesite of an angioplastic procedure (this high concentration would rapidlybe diluted out in the bloodstream), or a relatively longer exposure to alower concentration of CO. Examples of an artificial lungs aredescribed, e.g., in Hattler et al., Artif. Organs 18(11):806-812 (1994);and Golob et al., ASAIO J., 47(5):432-437 (2001). As used herein, theterm “intravessel carbon monoxide delivery device” refers to a catheterdevice, e.g., an artificial lung (or modified version thereof) capableof residing in a blood vessel for extended periods of time (includingindefinitely) and delivering CO to the patient systemically and/orlocally.

Normobaric Chamber

In certain instances, it would be desirable to expose the whole patientto CO. The patient would be inside an airtight chamber that would beflooded with CO at a level that does not endanger the patient, or at alevel that poses an acceptable risk without the risk of bystanders'being exposed. Upon completion of the exposure, the chamber could beflushed with air (e.g., 21% O₂, 79% N₂) and samples could be analyzed byCO analyzers to ensure no CO remains before allowing the patient to exitthe exposure system.

Systemic Delivery of Liquid CO Compositions

The present invention further contemplates that liquid CO compositionscan be created for systemic delivery to a patient, e.g., by intravenousor intraarterial infusion into a patient. For example, liquid COcompositions, such as CO-saturated Ringer's Solution, can be infusedinto a patient before, during, and/or after an angioplastic ortransplant procedure. Alternatively or in addition, CO-partially orcompletely saturated whole (or partial) blood can be infused into thepatient. The present invention also contemplates that agents capable ofdelivering doses of CO gas or liquids can be utilized (e.g., COreleasing gums, creams, ointments or patches).

Delivery of CO to Portions of the Vasculature

In Situ Treatment

Alternatively or in addition to systemic treatment, carbon monoxidecompositions can be applied directly to any portion of a patient'svasculature that has or is at risk for intimal hyperplasia and/orarteriosclerosis. A gaseous composition can be applied directly to aportion of a patient's vasculature, e.g., to an affected artery, by anymethod known in the art for administering gases into a patient'svasculature. For example, CO can be delivered to an artery before,during, and/or after an angioplastic (e.g., balloon angioplasty) orsurgical (e.g., transplant) procedure through a device similar to theintravenous artificial lung described above. As another example, anydevice used for performing an angioplastic procedure can be modified toadminister CO to a patient's vasculature through the instrument whileangioplasty is being performed. Such devices are discussed in furtherdetail below.

Liquid CO compositions can also be applied directly to a portion of apatient's vasculature. Liquid CO compositions can be administered by anymethod known in the art for administering liquids to the vasculature ofa patient. For example, a liquid CO composition can be administered to aspecific vein (e.g., by intravenous injection) or artery (e.g., byintraarterial injection) before, during, and/or after a procedure. Asanother example, as described above, any instrument used in angioplasticprocedures can be modified to administer to a vein or artery a liquid COcomposition while an angioplastic procedure is being performed.

Ex Vivo Treatment

The present invention further contemplates use of CO compositions toprevent or reduce intimal hyperplasia and/or arteriosclerosis intransplanted vasculature, e.g., individual blood vessels (e.g., vein oraortic transplants) or blood vessels that remain associated with atransplantable organ (e.g., kidney, liver, heart, or lung). Alternativeor in addition to the in situ exposures described above, exposure ofvasculature to CO compositions can occur ex vivo. For example, prior totransplanting individual blood vessels or an organ with its associatedvasculature into a recipient patient, the vasculature may be exposed toan atmosphere comprising carbon monoxide gas, to a liquid carbonmonoxide composition, e.g., a liquid perfusate, storage solution, orwash solution having carbon monoxide dissolved therein, or both.

Exposure of vasculature to gaseous CO compositions ex vivo can beperformed in any chamber or area suitable for creating an atmospherethat includes appropriate levels of CO gas. Such chambers include, forexample, incubators and chambers built for the purpose of accommodatingan organ in a preservation solution. As another example, an appropriatechamber may be a chamber wherein only the gases fed into the chamber arepresent in the internal atmosphere, such that the concentration ofcarbon monoxide can be established and maintained at a givenconcentration and purity, e.g., where the chamber is airtight. Forexample, a CO₂ incubator may be used to expose vasculature to a carbonmonoxide composition, wherein carbon monoxide gas is supplied in acontinuous flow from a vessel that contains the gas.

Exposure of vasculature to liquid CO compositions ex vivo may beperformed in any chamber or space having sufficient volume forsubmerging the vasculature, completely or partially, in a liquid COcomposition. Vasculature can also be exposed to such compositions byplacing the vasculature in any suitable container, and causing a liquidCO composition to “wash over” or through the vasculature, such that thevasculature is exposed to a continuous flow of the CO composition. Asyet another example, the vasculature may be submerged in a medium orsolution that does not include CO, and placed in a chamber such that themedium or solution can be made into a CO composition via exposure to aCO-containing atmosphere as described herein. As still another example,the vasculature may be submerged in a liquid that does not include CO,and CO can be “bubbled” into the liquid.

Devices

The present invention contemplates administering CO to a patient'svasculature using a device that is capable of being used for bothperforming an angioplasty procedure and administering CO to a patient'svasculature. CO can be administered through and/or by the instrument, orby a CO-delivering coating thereon, while angioplasty is being performed(e.g., immediately before, during, and/or immediately after angioplastyis performed). Such devices include devices used for balloon angioplasty(“balloon angioplasty devices”), laser angioplasty (“laser angioplastydevices”), and devices used for atherectomy (“atherectomy devices”),e.g., extraction atherectomy; directional atherectomy; rotationalatherectomy; and stents. As used herein, an “angioplasty device” is anydevice that can be used to perform angioplasty on a patient.

Referring to FIGS. 13A to 13D, examples of a catheter device with aninflatable member (e.g., a balloon) designed to administer CO (e.g., aliquid or gaseous CO composition) to a patient during angioplasty areshown. In FIG. 13A, the catheter 1303 is shown in position within astenotic region 1302 of blood vessel 1301. The inflatable member 1304 isshown in the deflated state. The inflatable member includes at least oneaperture 1305, through which CO can be administered to the vessel duringthe procedure. CO can be provided to the inflatable member (e.g., toinflate the inflatable member) from a reservoir (not shown) containingCO, e.g., a pharmaceutical composition comprising carbon monoxide. Thedevice can optionally be guided to the intended site by a guide wire1306.

In FIG. 13B, the catheter 1303 is shown with the inflatable member 1304in an inflated state. CO can be administered through the aperture(s)1305 to the vessel 1301. In one embodiment, the inflatable member isinflated with CO, or a mixture of gases including CO, such that anamount of the CO sufficient to treat intimal hyperplasia flows out ofthe aperture(s) 1305 and is delivered to the blood vessel 1301 duringand/or after inflation of the inflatable member 1304.

FIG. 13C illustrates another embodiment of the catheter device, whereinthe catheter 1303 includes at least one lumen 1307 for delivering CO tothe blood vessel 1301 during the angioplasty procedure. Still anotherembodiment is shown in FIG. 13D, wherein a central lumin 1307 deliversCO to a plurality of sites in the blood vessel 1301. A reservoircontaining CO (not shown) can be connected to the lumin 1307, such thata dose of CO is administered from the reservoir through the lumin 1307to the blood vessel.

Alternatively or in addition, the inflatable member 1304 can be coatedwith a CO-releasing coating, e.g., a hydrogel containing a COcomposition, such that CO is delivered to the stenotic region 1302,e.g., upon contact with the inflatable member 1304.

Referring to FIGS. 14A to 14B, an example of a stent designed toadminister CO to a patient is shown. The term “stent” is anart-recognized term and refers to a mesh tube, typically made of wire,used to maintain a blood vessel in an open position, e.g., a bloodvessel that has recently been remodeled during angioplasty. In FIG. 14A,a stent 1402 is shown in a collapsed state. The stent covers a ballooncatheter 1404, shown in FIG. 14A in a deflated state. In FIG. 14B, thestent 1402 and balloon catheter 1404 are shown in an expanded/inflatedstate. Upon inflation of the balloon catheter 1404, the stent 1402expands, locks in place, and forms a scaffold as shown in FIG. 14B,thereby holding the blood vessel in an open position. In one embodimentof the present invention, the stent 1402 is coated with a CO-releasingcoating, e.g., a hydrogel that releases CO, such that an amount of COsufficient to treat intimal hyperplasia is delivered to the blood vesselfor an appropriate amount of time, e.g., for as long as the stentremains in place.

Referring to FIG. 15, a catheter 1502 with two inflatable members 1504is shown. The inflatable members 1504 can be used to isolate a stenoticregion 1506, such that CO can be administered to the stenotic region1506 between the inflatable members 1504. The catheter 1502 is insertedinto the blood vessel 1508 prior to inflation of the inflatable members1504. The inflatable members 1504 are then inflated using aninflation/deflation tube 1510 housed within the catheter 1502. Theinflatable members 1504 in their inflated state obstruct the flow ofblood to the region of the blood vessel undergoing treatment. Intakeducts 1512 on the proximal end of the catheter allow blood to flow intoand through the catheter 1502 to outlet ducts 1514 located on the distalend of the catheter. This allows the blood to continue flowing to therest of the artery 1508 while the local site of the artery 1506 istreated. CO can be introduced to the isolated region through anadministering supply tube 1516. The inflated inflatable members 1504provide an isolated treatment area within which appropriate levels of COcan be administered to the vessel. In addition, fiber probes (not shown)can be secured to the casing of the catheter 1502, and the site can beexposed to electromagnetic radiation through the fiber probes. CO can beadministered to the site before, during, and/or after treatment of thesite with electromagnetic radiation.

Referring to FIG. 16, an example of an instrument capable ofadministering CO to a patient while performing atherectomy is shown.Atherectomy involves cutting away and removing plaque 1602 from bloodvessel walls 1604. The catheter 1606 is positioned within the artery1604. A flexible guide 1608 is used to move the instrument through theregion of treatment 1610. Rotating cutting blades 1612 are then extendedbeyond the catheter 1606. The rotating cutting blades 1612 follow theflexible guide 1608 and cut through the plaque 1602. The rotatingcutting blades 1612 draw the removed particles of plaque into andtowards the proximal end of the catheter 1606. CO can be introduced tothe treated region through an administering tube 1614 within thecatheter 1606. An administering tube 1614 can be secured to the guide1608. CO can be administered through the catheter 1606 through a leastone pore 1616 on the distal end of the administering tube 1614.Alternatively or in addition to supplying CO through an administeringtube 1614, ducts 1620 and 1622 can be housed within the walls of thecutting blades 1612. Carbon monoxide can be supplied through an outletduct 1620. At the conclusion of treatment an inlet duct 1622 can removethe CO. CO can be administered before, during, and/or after removal ofplaques.

In addition to the above, a skilled practitioner will appreciate thatany device known in the art for performing angioplasty procedures can bemodified to administer CO to a patient's vasculature during use.Examples of such devices can be found, e.g., in U.S. Pat. Nos.6,409,716, 5,985,307, 6,508,787, 5,709,875 and 6,450,989. Further, askilled practitioner will recognize that any such devices can be coatedwith a CO-delivering agent, e.g., an oil, ointment or hydrogel, capableof releasing effective doses of CO, such that the CO is delivered toblood vessel upon contact with the instrument/coating.

Use of Hemoxygenase-1 and Other Compounds

Also contemplated by the present invention is the induction orexpression of hemeoxygenase-1 (HO-1) in conjunction with administrationof carbon monoxide. HO-1 can be provided to a patient by inducing orexpressing HO-1 in the patient, or by administering exogenous HO-1directly to the patient. As used herein, the term “induce(d)” means tocause increased production of a protein, e.g., HO-1, in isolated cellsor the cells of a tissue, organ or animal using the cells' ownendogenous (e.g., non-recombinant) gene that encodes the protein.

HO-1 can be induced in a patient by any method known in the art. Forexample, production of HO-1 can be induced by hemin, by ironprotoporphyrin, or by cobalt protoporphyrin. A variety of non-hemeagents including heavy metals, cytokines, hormones, nitric oxide, COCl₂,endotoxin and heat shock are also strong inducers of HO-1 expression(Otterbein et al., Am. J. Physiol. Lung Cell Mol. Physiol.279:L1029-L1037, 2000; Choi et al., Am. J. Respir. Cell Mol. Biol.15:9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37:517-554, 1997;and Tenhunen et al., J. Lab. Clin. Med. 75:410-421, 1970). HO-1 is alsohighly induced by a variety of agents and conditions that createoxidative stress, including hydrogen peroxide, glutathione depletors, UVirradiation and hyperoxia (Choi et al., Am. J. Respir. Cell Mol. Biol.15: 9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37:517-554, 1997;and Keyse et al., Proc. Natl. Acad. Sci. USA 86:99-103, 1989). A“pharmaceutical composition comprising an inducer of HO-1” means apharmaceutical composition containing any agent capable of inducing HO-1in a patient, e.g., any of the agents described above, e.g., hemin, ironprotoporphyrin, and/or cobalt protoporphyrin.

HO-1 expression in a cell can be increased via gene transfer. As usedherein, the term “express(ed)” means to cause increased production of aprotein, e.g., HO-1 or ferritin, in isolated cells or the cells of atissue, organ or animal using an exogenously administered gene (e.g., arecombinant gene). The HO-1 or ferritin is preferably of the samespecies (e.g., human, mouse, rat, etc.) as the recipient, in order tominimize any immune reaction. Expression could be driven by aconstitutive promoter (e.g., cytomegalovirus promoters) or atissue-specific promoter (e.g., milk whey promoter for mammary cells oralbumin promoter for liver cells). An appropriate gene therapy vector(e.g., retrovirus, adenovirus, adeno associated virus (AAV), pox (e.g.,vaccinia) virus, human immunodeficiency virus (HIV), the minute virus ofmice, hepatitis B virus, influenza virus, Herpes Simplex Virus-1, andlentivirus) encoding HO-1 or ferritin would be administered to thepatient orally, by inhalation, or by injection at a location appropriatefor treatment intimal hyperplasia. Similarly, plasmid vectors encodingHO-1 or apo-ferritin can be administered, e.g., as naked DNA, inliposomes, or in microparticles.

Further, exogenous HO-1 protein can be directly administered to apatient by any method known in the art. Exogenous HO-1 can be directlyadministered in addition to, or as an alternative, to the induction orexpression of HO-1 in the patient as described above. The HO-1 proteincan be delivered to a patient, for example, in liposomes, and/or as afusion protein, e.g., as a TAT-fusion protein (see, e.g., Becker-Hapaket al., Methods 24:247-256, 2001).

Alternatively or in addition, any of the products of metabolism by HO-1,e.g., bilirubin, biliverdin, iron, and/or ferritin, can be administeredto a patient in conjunction with, or instead of, carbon monoxide inorder to prevent or treat intimal hyperplasia. Further, the presentinvention contemplates that iron-binding molecules other than ferritin,e.g., desferoxamine (DFO), iron dextran, and/or apoferritin, can beadministered to the patient. Further still, the present inventioncontemplates that enzymes (e.g., biliverdin reductase) that catalyze thebreakdown any of these products can be inhibited to create/enhance thedesired effect.

The present invention contemplates that compounds that release CO intothe body after administration of the compound (e.g., CO-releasingcompounds), e.g., dimanganese decacarbonyl, tricarbonyldichlororuthenium(II) dimer, and methylene chloride (e.g., at a dose of between 400 to600 mg/kg, e.g., about 500 mg/kg) can also be used in the methods of thepresent invention, as can carboxyhemoglobin and CO-donating hemoglobinsubstitutes, and.

Administration any of the above can be administered to a patient in anyway, e.g., by oral, intravenous, or intraarterial administration. Any ofthe above compounds can be administered to the patient locally and/orsystemically, and in any combination.

The invention is illustrated in part by the following examples, whichare not to be taken as limiting the invention in any way.

EXAMPLE 1 CO Suppresses Arteriosclerosis, Development of IntimalHyperplasia and SMC Proliferation

Animals. Male (250-350 g) Brown Norway rats (RT1^(n)) were used asaortic graft donors and male (250-350 g) Lewis rats (RT1₁) asrecipients. Dawley (400-450 g) rats were used in the balloon injurymodel. Adult male C57BL/6, C57/S129, p21^(−/−) and p53^(−/−) null micewere purchased from Jackson Laboratory (Bar Harbor, Me.). mkk3^((−/−))null mice were generated as described Lu et al. (EMBO. 18:1845-1857(1999)). The inos^(−/−) and enos^(−/−) mice were bred at the Universityof Pittsburgh.Aortic transplant model. Aortic transplantation was performed asdescribed in Shimizu et al. (Nat. Med. 7:738-741 (2001)). Briefly, 3 to4 cm of descending aorta was harvested from the donor and implantedbetween the renal arteries and the aortic bifurcation of the recipient.Both edges of the native abdominal aorta were ligated.Balloon injury model. Balloon angioplasty was carried out as describedin Murakami et al. (Atherosclerosis 157:361-368 (2001)). Briefly, a 2Fr. arterial embolectomy catheter (Baxter, Chicago, Ill.) was insertedinto the common carotid artery, and injury was created by inflating theballoon to 5 atmospheres of pressure for 5 minutes. The arteries wereflushed and the external carotid artery was ligated, ensuring return ofblood flow through the common and internal carotid arteries. Injury ofthe vessel wall and subsequent pathological analysis was made in amanner that was blinded to the treatment group.CO exposure. CO was delivered to animals as described in Otterbein etal. (Nat. Med. 6:422-428 (2000)). Graft donors and recipients wereexposed to CO (250 ppm) for two days before transplantation and for 56days immediately following transplantation. In the balloon injury model,rats received either no pretreatment or were exposed to CO (250 ppm) forone hour prior to injury. Following surgery rats were housed in room airfor two weeks.Cells. Primary mouse and rat smooth muscle cells (SMC) were isolated andcultured as described in Laubach et al. (Proc Natl Acad Sci USA92:10688-9 (1995)). Mouse SMC isolated from HO-1^(−/−) mice wereobtained as described in Duckers et al. (Nat. Med. 7:693-698 (2001)).Cell treatment and reagents. Guanylate cyclase inhibitor 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1 (ODQ; Calbiochem-Novabiochem, San Diego,Calif.; 10-100 μM) and p38 MAPK inhibitor pyridinyl imidazol SB203580(Calbiochem; 5-20 μM) were dissolved in DMSO. The cGMP analogue8-bromo-cGMP sodium salt (8-Br-cGMP; Sigma-Aldrich, St. Louis, Mo.;10-100 μM) and the PKG inhibitor (10-100 μM; Alexis Biochemicals) weredissolved in water.Cell counts and [³H] thymidine incorporation. Rat and mouse SMC wereisolated and cultured as described in Peyton et al. (Blood 99:4443-4448(2002)). Proliferation assays were carried out as described in Petkovaet al. (J Biol. Chem. 276:7932-7936 (2001)). For [³H] thymidineincorporation studies, cells were serum-starved overnight and thenstimulated with 10% serum containing 5 μCi/ml [³H] thymidine (NewEngland Nuclear, Boston Mass.). [³H]thymidine incorporation was measuredby scintillation spectroscopy and presented as mean counts/min/well.Histomorphometric analysis. Aortic grafts and carotid arteries wereharvested at 56 and 14 days respectively. Vessels were fixed, embedded,and serially sectioned (5μ) in toto. Every third slide was stained withHematoxylin and Eosin (H&E) for histomorphometric analyses. In bothmodels, one or two images per slide at a resolution of 1520×1080 pixelswere captured at a magnification of 25× with a Zeiss microscope(Axioskop, Iowa City, Iowa), RT color SPOT (Diagnostic Instrument, Inc.,Saint Joseph, Mich.) and Windows NT (Compaq Computer) using AdobePhotoshop version 5.5 software. Areas from eight to ten captured imageswere calculated using digital imaging software as number of pixelscorresponding to those areas. Twenty-four to forty-eight sections fromeach group were statistically analyzed with SPSS software version 10.Immunostaining and cell population histomorphometric analysis. Graftswere harvested 56 days after transplantation. Rat leukocyte populationswere detected using anti-rat leukocyte common antigen (LCA, CD45; OX-1)(Serotec, Harlan Bioproducts, Indianapolis, Ind.); CD3 (G4.18), CD4(OX-35), CD8 (OX-8) macrophage (CD68, ED-1), ICAM-1 (CD54; 1A29), andmajor histocompatibility class II (OX-6) were all obtained from BectonDickinson Biosciences, (San Diego, Calif.). Anti-PAI-1 mAb was obtainedfrom America Diagnostica (Greenwich, Conn.). Eight to ten images werecaptured from each transplanted aorta and analyzed as detailed above.Cell extracts and Western Blot Analysis. Cellular protein extracts wereelectrophoresed (10-12.5% polyacrylamide gels) and transferred ontonitrocellulose (BioRad, Hercules, Calif.). Total and phosphorylatedforms of ERK, JNK and p38 MAPK as well as ATF-2 were detected usingrabbit polyclonal antibodies (Cell Signaling Technologies, Beverly,Mass.). Anti α-actin (Sigma; St. Louis, Mo.). p21^(Cip1) was detectedusing a rabbit polyclonal antibody (Santa Cruz Biotech, Santa Cruz,Calif.). Primary antibodies were detected as described in supplementarymethods.Statistical Analysis. The significance of difference was determinedusing analysis of variance (ANOVA).Nitric Oxide (NO) exposure. Rats were exposed to 250 ppm or 500 ppm NOfor 1 hour. NO gas (1% in N2) was mixed with air in the same exposureapparatus as used in the CO experiments. Concentrations in the chamberwere monitored with an NO analyzer (Interscan). Following exposure,balloon angioplasty was carried out as described above. Control animalswere exposed to air. The surgeon inflicting the balloon injury wasblinded to the rats being manipulated. Analyses of carotid arteries wasperformed 2 weeks after the procedure as described above.Mouse arterial injury. The dissection was similar to that described byLindner et al. (Circ Res. 73:792-796 (1993)), and was performed using a0.018 inch guide wire (Cook, Bloomington, Ind.), inserted through anexternal carotid arteriotomy into the common carotid, rotated 360degrees three times and removed a total of three consecutive times.cGMP immunoassays. Cellular levels of cGMP were quantified using an EIA(Biomol, Plymouth Meeting, Pa.). SMC were incubated in the presence orabsence of CO (250 ppm) and cell lysates were analyzed for cGMP content,as suggested by the vendor.Cell Counts. Cells were seeded at 5×10³ cells/well and culturedovernight in high glucose DMEM containing 10% FCS, penicillin, andgentamicin (Life Technologies). Cells were serum starved for anadditional 48 hours (0% serum) and where indicated exposed to CO (250ppm for rat and mouse SMC) before induction of cell proliferation (10%FCS; Life Technology). Cells were counted daily using a Neubauerhemocytometer. Viability was assessed with trypan blue.Recombinant adenovirus. Recombinant β-galactosidase adenovirus wasobtained from the University of Texas Southwest Medical Center, Dallas,Tex. Recombinant HO-1 adenovirus expressing the rat HO-1 cDNA has beendescribed in Brouard et al. (J Exp Med. 192:1015-1026 (2000)). Rat SMCwere infected with a multiplicity of infection (MOI) of 400 plaqueforming units per cell (PFU/cell), as described in Brouard et al. (Id.).Flow Cytometry. Rat aortic SMC were harvested by trypsin digestion(0.025% Trypsin/0.01% EDTA) (Life Technology), washed in phosphatebuffered saline (PBS, pH 7.2) with 0.5% bovine serum albumin (BSA;Sigma-Aldrich Co), and incubated with Propidium iodide (1 μg/ml, 1 h,RT). Fluorescent labeling was evaluated using a FACsort equipped withCell Quest Software (Becton Dickinson, Palo Alto, Calif.). Experimentswere carried out in triplicate.Histomorphometric analyses. In the transplant model, grafts wereharvested 56 days after transplantation. Aortas were fixed in 10%formalin, embedded in paraffin and serially sectioned (5μ) in toto. Tensamples from every three sections were placed per slide in a total ofabout twenty-four to thirty slides. Every third slide was stained withHematoxylin and Eosin (H&E) for histomorphometric analysis. In theballoon injury model, animals were euthanized 14 days following injuryand arteries were collected for morphometric analysis. Rat carotidarteries were perfused and fixed in situ with PBS and paraformaldehyde(2%). Vessels were fixed for 2 hours in 2% paraformaldehyde at 4° C. andcryoprotected in 30% sucrose overnight at 4° C. Vessels werequick-frozen in 2-methylbutane and 7 μm cryosections were cut.Primary Antibody Detection for Immunoblotting. Primary antibodies weredetected using horseradish peroxidase conjugated anti-rabbit IgGsecondary antibodies (Pierce, Rockford, Ill., USA). Peroxidase wasvisualized using the Enhanced ChemiLuminescence assay (Amersham LifeScience Inc., Arlington Heights, Ill., USA), according to manufacturer'sinstructions and stored in the form of photoradiographs (BiomaxTMMS,Eastman Kodak, Rochester, N.Y.). Where indicated, membranes werestripped (62.5 mM Tris.HCl pH 6.8, 2% SDS and 100 mM β-mercaptoethanol,30 minutes, 50° C.). Phosphorylated p38 were normalized to the totalamount of p38, detected in the same membrane.Miscellaneous Reagents. Mouse iNOS and eNOS were detected using rabbitanti-mouse polyclonal antibodies against iNOS and eNOS (BectonDickinson, Biosciences, San Diego, Calif.).

CO Suppresses the Development of Transplant-Associated Arteriosclerosis.

FIGS. 1A-1I illustrate that CO treatment suppresses intimal hyperplasianormally associated with chronic graft rejection. FIGS. 1A-1F arephotomicrographs of samples of various aortic grafts 56 days aftertransplantation. To generate these data, Brown Norway aortas weretransplanted into Brown Norway rats (FIGS. 1A and 1D), Lewis ratsexposed to air (FIGS. 1B and 1E), and Lewis rats exposed to CO (250 ppm;FIGS. 1C and 1F). Samples were harvested 56 days after transplantationand stained by a modified elastic tissue-masson trichrome (elastic;FIGS. 1A-1C) or by hematoxylin and eosin (H&E; FIGS. 1D-1E). Elasticstainings are magnified 50× (FIGS. 1A-1C) and H&E stainings aremagnified 200× (FIGS. 1D-1E). Samples shown are representative of 3-6animals analyzed per group. FIGS. 1G-1I are bar graphs illustrating themean (±standard deviation) relative areas corresponding to the intimaand media regions, calculated from samples harvested from Brown Norwayaortas transplanted into Brown Norway rats (Syng.; n=6), Lewis ratsexposed to air (Allo.; n=6) or CO (250 ppm) (Allo.+CO; n=3). *P<0.001versus Allo.+CO.

Brown Norway aortic segments transplanted into Lewis rats developedarteriosclerotic lesions consistent with chronic graft rejection (FIGS.1B and 1E). The lesions appeared 20-30 days after transplantation butwere significantly more pronounced by 50-60 days; all analyses werecarried out 56 days following transplantation. The lesions werecharacterized by intimal hyperplasia, loss of medial SMC, and leukocyteaccumulation in the adventitia (FIGS. 1B and 1E). These characteristicswere not observed in vessels of the recipient, and the lesions were notobserved in syngeneic grafts (FIGS. 1A and 1D). Intimal hyperplasia wassignificantly (p<0.001) inhibited (61.4±12.9% reduction versus control)in aortas transplanted into recipients exposed to CO (250 ppm)immediately after transplantation (and for 56 days thereafter), ascompared to those transplanted into air-exposed recipients (FIGS. 1C,1F, 1G, 1H, an 1I).

FIGS. 2A-C are bar graphs illustrating that CO suppresses graftinfiltration by activated leukocytes. To generate the data in FIGS.2A-2C, immunocytochemical analyses were performed on aortic grafts 56days after transplantation. Brown Norway rat aortas were transplantedinto Brown Norway rats (syngeneic), untreated Lewis rats (allogeneic) orLewis rats exposed to CO (250-1000 ppm). Samples were harvested 56 daysafter transplantation. FIG. 2A illustrates the mean (±standard deviation(n=3-6)) number of nuclei in the adventitia from Brown Norway aortastransplanted into Brown Norway recipients (Syngeneic), untreated Lewisrecipients (Allogeneic), and Lewis recipients exposed to variousconcentrations of CO(CO 250 ppm, CO 500 ppm, and CO 750-1000 ppm)(*=P<0.001 versus Allo.). FIG. 2B illustrates the mean (±standarddeviation (n=6)) number of CD45 (*P<0.002 versus Allo.), CD68 (Mø/ED1;*P<0.001 versus Allo.), MHC II, and CD54 (ICAM-1) positive cells(*P<0.001 versus Allo.) in the adventitia from Brown Norway rat aortastransplanted into Brown Norway rat recipients (Syng.), untreated Lewisrat recipients (Allo.), and Lewis rat recipients exposed to CO(Allo.+CO). FIG. 2C illustrates the mean (±standard deviation (n=6))number of CD3, CD4, and CD8 positive cells (*P<0.02, 0.001, 0.096respectively versus Allo.) in the adventitia from Brown Norway aortastransplanted into Brown Norway recipients (Syng.), untreated Lewisrecipients (Allo.), and Lewis recipients exposed to CO(CO 250 ppm).

Accumulation of leukocytes in the adventitia of transplanted aortas wasinhibited in CO-exposed recipients (see FIGS. 2A-2C). Leukocyteaccumulation was not observed in syngeneic grafts. The ability of CO tosuppress graft infiltration by activated leukocytes (CD45+) was dosedependent with increasing levels of CO (250-1000 ppm) resulting indecreased leukocyte infiltration; a maximal effect was observed at 700to 1000 ppm of CO (52±20% inhibition versus air treated controls FIG.2A). CO significantly suppressed the accumulation of CD45+/CD68+monocyte/macrophages (Mø) (65±24% inhibition versus air treatedcontrols) as well as CD45+/CD3+ T cells (57±22% inhibition versus airtreated controls), including both CD4⁺ (“helper”) and CD8⁺ (“cytotoxic”)cells (FIG. 2C). CO also inhibited expression of pro-inflammatory genesassociated with Mø activation including the major histocompatibilityclass II (MHC II) antigens and the intracellular adhesion molecule 1(CD54/ICAM-1) (FIG. 2B).

CO Suppresses Development of Intimal Hyperplasia after Balloon Injury.

FIGS. 3A-3G illustrate that CO suppresses the development of vascularlesions associated with balloon injury. FIGS. 3A-3D are photomicrographs(10× magnification) of immunocytochemically stained carotid arteriesanalyzed 14 days after balloon angioplasty. To generate the data inFIGS. 3A-3D, rats were exposed to room air (FIGS. 3A and 3C) or to CO (1hour; 250 ppm; FIGS. 3B and 3D) prior to balloon injury. All animalswere exposed to room air following balloon injury. Two weeks afterballoon injury, samples were stained with hemotoxylin and eosin (H&E).Samples from room air (FIGS. 3A and 3C) and CO pretreated rats (FIGS. 3Band 3D) are shown. FIGS. 3E, 3F, and 3G are bar graphs illustrating themean (±standard deviation (n=8; *P<0.001 versus control)) relative areasof the intima and media regions of samples analyzed in FIGS. 3A-3D.

Rat carotid arteries developed intimal hyperplasia 14 days after ballooninjury (FIGS. 3A and 3C; and FIGS. 3E-3G). Intimal hyperplasia in ratsexposed to CO (250 ppm) for one hour prior to balloon injury (afterwhich CO exposure was discontinued) was suppressed by 74±8% as comparedto control animals exposed to air (n=8-10; p<0.001) (FIGS. 3B and 3D andFIGS. 3E-3G).

CO Suppresses SMC Proliferation.

FIGS. 4A-4F illustrate that CO blocks SMC proliferation and thatp21^(Cip1) is involved in the anti-proliferative effect of CO in vitro.FIG. 4A is a line graph illustrating proliferation of rat SMC that werenon-transduced (∘; Medium), or transduced with Lac.Z (□; LacZ Rec. Ad.)or HO-1 (; HO-1 rec. Ad.) recombinant adenovirus. Results shown aremean ±standard deviation of n=3 wells per group (*P<0.001 versus LacZand non-transduced). FIG. 4B is a line graph illustrating proliferationof rat SMC growth in the presence or absence of CO (; 1000 ppm).Results shown are mean ±standard deviation (n=3 wells per group; P<0.001versus air, □). FIG. 4C is a bar graph illustrating proliferation ofSMC₁ isolated from wild type (WT) or HO-1 deficient (ho-1^(−/−)) mice inthe presence and absence of CO (250 ppm) (n=6 wells/group; #P<0.006versus air, *P<0.001). FIG. 4D is a Western blot illustrating p21 andβ-actin protein expression in mouse SMC following exposure to CO (250ppm). FIG. 4E is a bar graph illustrating proliferation of mouse SMCisolated from wild type (wt), p21^(Cip1) (p21−/−) or p53 (p53−/−)deficient mice. Gray bars indicate cells exposed to room air and blackbars indicate cells exposed to CO (250 ppm). Results shown are the mean±standard deviation (n=3*P<0.001 versus air) in one out of 6 independentexperiments. FIG. 4F is a bar graph illustrating that endogenousexpression of p21^(Cip1) plays a critical role in controlling the extentof intimal hyperplasia following arterial injury in mice. Wild type(C57/B16/S129) or p21−/− mice were exposed to room air or CO (1 hour;250 ppm) before carotid artery injury and exposed to room airthereafter. Samples were harvested and analyzed two weeks after injury.Mean ±standard deviation (n=4) of the ratio between the relative area ofthe intima and media is expressed in arbitrary units (*P<0.001 versusair).

FIGS. 7A-7B further illustrate that CO blocks SMC proliferation. FIG. 7Ais a cell cycle analysis of rat aortic SMC in standard incubation (Air)versus CO (250 ppm) after 24 hours of treatment. Results shown arerepresentative of 3 independent experiments. FIG. 7B is a line graphillustrating SMC proliferation in the presence (▪; CO, 250 ppm) orabsence (□; air) of CO for 6 days. SMC were serum stimulated on day 3 ofthe experiment. Proliferation increased in CO-treated SMC after COexposure was discontinued on day 6. Results shown are representative of3 independent experiments, each performed in triplicate (*P<0.01 vsAir).

FIG. 8 is a bar graph illustrating that CO blocks SMC proliferation fromwild type (wt) and p53^(−/−) mice but not from p21^(−/−) mice. Thefigure presents the results of a [³H]thymidine incorporation assay usedto assess cellular proliferation (counts per minute; cpm) in wild type(wt), p21^(−/−, and p)53^(−/−) mouse SMCs exposed to air (gray bars) orto CO (250 ppm; black bars) for 24 hours. Results shown are the mean±standard deviation (n=3; *P<0.05 versus air).

FIGS. 9A-9G illustrate that CO suppresses the development of vascularlesions associated with wire injury in wild type (C57/B16/S129) andp21^(−/−) mice. FIGS. 9A-9D are photomicrographs (20× magnification) ofimmunocytochemically stained carotid arteries 14 days following wireinjury. Mice were exposed to room air (FIGS. 9A and 9C) or CO (1 hour;250 ppm; FIGS. 9B and 9D) prior to wire injury. All animals were exposedto room air following injury. Two weeks after wire injury, samples werestained with hemotoxylin and eosin (H&E). Samples from wild type (FIGS.9A and 9B) and p21^(−/−) (FIGS. 9C and 9D) mice are shown. FIGS. 9E, 9F,and 9G are bar graphs illustrating the Mean (±standard deviation(n=4*P<0.001 versus control)) relative areas corresponding to the intimaand media regions, as well as intima:media ratios, from thephotomicrographs shown in FIGS. 9A-9D.

CO Suppresses SMC Proliferation In Vitro

An in vitro system was used to evaluate SMC growth in the presence orabsence of CO to delineate the mechanism by which CO inhibited intimalhyperplasia. Serum starved SMC proliferated upon re-addition of serum toculture medium (FIG. 7B); control SMC not exposed to serum exhibitedminimal proliferation during the five days of the experiment. Expressionof a HO-1 recombinant adenovirus or exposure to CO suppressed SMCproliferation (FIGS. 4A and 4B). In a similar system, “scavenging” of COby hemoglobin suppressed the anti-proliferative effect of HO-1,suggesting that CO generated by HO-1 likely accounts for theanti-proliferative effect of HO-1. Cell cycle analysis revealed that SMCtreated with CO accumulated in the G0/G1 phase (FIG. 7A). SMC from micethat lack HO-1 (ho-1^(−/−)) proliferated significantly more rapidly whenexposed to serum than did SMC from wild type mice (FIG. 4C), indicatingthat endogenous HO-1 expression in SMC exposed to serum suppressessmooth muscle cell proliferation. CO significantly inhibitedproliferation of SMC from ho-1^(−/−) mice, suggesting that CO accountsin large measure for the anti-proliferative action of HO-1. Inhibitionof SMC proliferation was not associated with cell death as assessed bytrypan blue and propidium iodide exclusion analyses. Theanti-proliferative effects of CO were reversible, as cessation of COexposure allowed SMC to begin to proliferate again (FIG. 7B).

The In Vitro Anti-Proliferative Effect of CO Depends on p21^(Cip1).

SMC exposed to CO up-regulated p21^(Cip1) protein expression (FIG. 4D),similar to SMC that overexpressed HO-1. This effect was transitory inthat p21^(Cip1) expression increased significantly by 4 hours, wasmaximal by 16 hours and returned to basal levels at 24 hours after COexposure (FIG. 4D). Proliferation of SMC from p21^(Cip1) deficient mice(p21^(−/−)) was not suppressed by CO (FIG. 4E), indicating thatp21^(Cip1) expression is required for the anti-proliferative effect ofCO. Despite the established role of p53 in regulation of p21^(Cip1)expression, CO induced p21^(Cip1) expression and inhibited proliferationof SMC derived from p53^(−/−) or from wild type mice to a similar extent(FIG. 4E and FIG. 8). Thus, the anti-proliferative effect of CO in vitrois dependent on p21^(Cip1) expression, which does not involve p53.

Involvement of p21^(Cip1) in the In Vivo Anti-Proliferative Effect of CO

p21^(Cip1) is expressed following vascular wall injury, likelyfunctioning to regulate SMC proliferation and the development of intimalhyperplasia. The role of endogenous p21^(Cip1) expression on developmentof intimal hyperplasia following arterial injury in mice wasinvestigated. Intimal hyperplasia was three times more pronounced in airtreated p21^(−/−) mice than in wild-type mice (n=4; 314±41.8%, p<0.001),showing that endogenous expression of p21^(Cip1) plays a critical rolein controlling the extent of intimal hyperplasia following arterialinjury in mice (FIG. 4F). In contrast, one hour of CO (250 ppm)pre-treatment suppressed intimal hyperplasia in both wild-type(C57/S129) and p21^(−/−) (C57/S129) mice by 80.9±7.2% (n=4, p<0.001) and86.2±14% (n=4, p<0.001), respectively, versus air treated controls (FIG.4F; FIGS. 9A-9G). Thus, in this in vivo mouse model, CO suppressesintimal hyperplasia via a mechanism that is not dependent on theexpression of p21^(Cip1).

The Anti-Proliferative Effect of CO Requires Activation of GuanylateCyclase and Generation of cGMP, and is Exerted Via Activation of p38MAPK.

FIGS. 5A-5G illustrate that CO blocks SMC proliferation via generationof cGMP and activation of p38 MAPK. FIG. 5A is a bar graph illustratingthe mean (±standard deviation (n=3)) cellular cGMP content in mouse SMCexposed to air or CO (250 ppm for 8 h or 16 h). FIG. 5B is a bar graphillustrating the results of a [³H]thymidine incorporation assay used toassess cellular proliferation (counts per minute; cpm) in mouse SMCexposed to CO (250 ppm) in the presence or absence of the guanylatecyclase inhibitor ODQ. Results shown are the mean standard deviation(n=3; *P<0.05 versus air and CO/ODQ). FIG. 5C is a picture of a Westernblot of p21^(Cip1) mouse SMC exposed to the cGMP analogue 8Br-cGMP.Membranes were subsequently probed for β⁻-actin to assure equal loading.Results shown are representative of 3 independent experiments. FIG. 5Dis a bar graph illustrating [³H]thymidine incorporation of SMC isolatedfrom wild type (wt) and p21^(Cip1) (p21^(−/−)) deficient mice in thepresence and absence of the cGMP analogue 8Br-cGMP. Results shown arethe mean ±standard deviation (n=4; *P<0.05 versus air and 8BrcGMP). FIG.5E is a composite picture of a Western blot of phosphorylated p38 MAPK(p-p38), ATF-2 (p-ATF-2), JNK (p-JNK) and ERK (p-ERK) of SMC exposed toCO (250 ppm). Membranes were subsequently probed with an antibodyagainst total p38 MAPK, ATF-2, JNK and ERK to assure equal loading.Blots are representative of 3 independent experiments. FIG. 5F is a bargraph illustrating [³H]thymidine incorporation in mouse SMC exposed toCO (250 ppm) in the presence and absence of the p38 MAPK inhibitorSB203580. Results shown are the mean ±standard deviation from 4independent experiments (p<0.005; versus air and CO/SB treated cells).FIG. 5G is a composite picture of a Western blot of p21^(Cip1) frommouse SMC exposed to CO (250 ppm) in the presence and absence of the p38MAPK inhibitor SB203580 or DMSO, used as a vehicle. The same membranewas probed with an antibody against β-actin to assure equal loading.Results shown are representative of 3 independent experiments.

FIGS. 6A-6B illustrate that CO activates p38 MAPK through a mechanismthat requires the generation of cGMP. FIG. 6A is a composite picture ofa Western blot of phosphorylated p38 MAPK from mouse SMC exposed to thecGMP analogue 8BrcGMP. Membranes were subsequently probed with anantibody against total p38 to assure equal loading. The composite blotshown in FIG. 6A is representative of 3 independent experiments. FIG. 6Bis a bar graph illustrating [³H]thymidine incorporation in mouse SMCexposed to CO (250 ppm) in the presence and absence of the cGMP analog8-BrcGMP. Results shown are the mean ±standard deviation from 4independent experiments.

FIG. 10 is a bar graph illustrating that CO does not suppressproliferation in SMC from p21^(−/−) mice. The bar graph presents theresults of a [³H]thymidine incorporation assay to assess cellularproliferation (counts per minute; cpm) in SMC derived from p21^(−/−)mice treated with CO (250 ppm) in the presence and absence of the p38MAPK inhibitor SB203580 (SB).

Exposure of SMC to CO increased intracellular cGMP levels (FIG. 5A). Theability of CO to suppress SMC proliferation and to up-regulatep21^(Cip1) expression was impaired under inhibition of guanylate cyclaseactivity by 1H(1,2,4) Oxadiazolo(4,3-a) Quinoxalin-1 (ODQ) (FIG. 5B).The non-degradable cGMP analogue 8-Bromoguanosine 3′-5′-cyclicmonophosphate sodium salt (8-Br-cGMP) suppressed SMC proliferation (FIG.5D) and increased expression of p21^(Cip1) comparably to CO (FIG. 5C).Suppression of proliferation by 8-Br-cGMP was impaired in SMC derivedfrom p21^(−/−) mice (FIG. 5D). It thus appears that theanti-proliferative effect of CO is mediated via activation of guanylatecyclase, accumulation of cGMP and expression/activation of p21^(Cip1).Activation of cGMP dependent kinases (PKG α and β) seems to be requiredfor CO to suppress SMC proliferation since an inhibitor of PKG abrogatedthe anti-proliferative effect of CO.

Whether the anti-proliferative effect of CO in SMC involved theactivation of the p38 MAPK signal transduction pathway was investigated.CO activated p38 MAPK in SMC (FIG. 5E), as it does in endothelial cellsand monocytes/macrophages. p38 MAPK phosphorylation/activation peakedfour hours after exposure to CO and returned to basal levels thereafter(FIG. 5E). Exposure of SMC to CO was also associated with the activationof ATF-2, a transcription factor most often activated through the p38MAPK signal transduction pathway (FIG. 5E). Inhibition of p38 MAPKactivation by the pyridinyl imidazol SB203580, a selective inhibitor ofthe p38α and β isoforms, blocked the ability of CO to up-regulateexpression of p21^(Cip1) (FIG. 5G) and suppressed the anti-proliferativeeffect of CO (FIG. 5F). Additionally, CO did not inhibit proliferationin cells isolated from mice deficient in mitogen activated proteinkinase kinase 3 (mkk3^(−/−)), the upstream kinase that activates p38αand p38β. SB203580 did not modulate the effects of CO on proliferationof SMC from p21^(−/−) mice (FIG. 10). These data indicate that p38 MAPKactivation is critical for upregulation of p21^(Cip1) and inhibition ofSMC proliferation by CO. CO did not modulate activation ofextra-cellular regulated kinases 1 and 2 (ERK-1 and -2) (FIG. 5E),indicating that this signal transduction pathway is not involved in theanti-proliferative effect of CO. CO induced activation of jun-activatedkinases 1 and 2 (JNK-1 and -2) (FIG. 5E).

Given that CO induces the generation of cGMP (FIG. 5A) and activation ofp38 MAPK (FIG. 5E) in SMC, the interrelationship between these twosignal transduction pathways was investigated. Activation of p38 MAPK byCO was abolished when the generation of cGMP was blocked by ODQ.Exposure of SMC to 8-bromo-cGMP activated p38 MAPK (FIG. 6A) and theanti-proliferative effect of 8Br-cGMP was abrogated in the presence ofSB203580 (FIG. 6B). Consistent with these findings, the ability of8Br-cGMP to up-regulate the expression of p21^(Cip1) was suppressed bySB203580.

The Anti-Proliferative Effect of CO does not Require Expression ofNitric Oxide Synthases.

FIGS. 1A-11B illustrate that NO is not involved with theanti-proliferative effect of CO. FIG. 11A is a bar graph illustrating[³H]thymidine incorporation in mouse SMC isolated from wild type (wt)and enos^(−/−) and inos^(−/−) deficient mice in the presence and absenceof CO (250 ppm). Results shown are the mean ±standard deviation (n=6-8,*P<0.001 versus wt). FIG. 11B is a bar graph illustrating the meanintimal and media areas of carotid arteries from Sprague-Dawley ratsexposed to room air (white bars) or NO (Black bars; 1 hour; 250 ppm)before balloon injury of the carotid artery. All animals were exposed toroom air following injury. Two weeks after injury carotid arteries wereremoved, sectioned and stained with hematoxylin and eosin (H&E). Areascorresponding to the intimal and media regions were calculated. Resultsare the mean ±standard deviation from representative 40 sections takenfrom 3 rats per treatment group.

SMC exposed to CO (250 or 10,000 ppm) for varying amounts of time showedno induction of either NOS isoform by western blotting. SMC from micedeficient for the constitutive/endothelial isoform of nitric oxidesynthase (eNOS/NOS-3; nos-3^(−/−)) or the inducible isoform (iNOS/NOS-2;nos-2^(−/−)) showed a significantly greater uptake of thymidine whenexposed to serum as compared to SMC derived from wild type mice (FIG.11A). Exposure to CO significantly inhibited proliferation of SMCderived from wild type, nos-2^(−/−) or nos-3^(−/−) mice (51±12.9% and51±25% and 54±11% inhibition respectively; p<0.001 versus air treatedcontrols) (FIG. 11A). These data indicate that the anti-proliferativeeffect of CO can occur in the absence of iNOS or eNOS. Similarly, undersimilar conditions to the ones used for CO (250 ppm; one hourpre-treatment), NO did not modulate intimal thickening triggered byballoon injury. NO administered at 500 ppm was lethal.

CO does not Inhibit the Expression of the Plasminogen ActivatorInhibitor Type 1 (PAI-1) in SMC.

FIGS. 12A, 12B and 12C illustrate that CO increases PAI-1 proteinexpression levels. FIG. 12A is a composite picture of a Western blotanalysis for PAI-1 in SMC treated with and without CO (250 ppm) for 24or 48 hours. Whole cell lysates from rat liver homogenates treated withand without endotoxin (LPS) were used as controls. α-actin was used toassay protein loading. FIG. 12B is a composite picture of a Western blotanalysis for PAI-1 in untransplanted (control), transplanted(Allo.+Air), and CO-treated transplanted (Allo.+CO) aortas after 56days. FIG. 12C is the Commassie blue-stained polyacrylamide gel used tocreate the Western blot of FIG. 12B, and illustrates that the sameamount of protein is loaded in each lane.

CO suppresses expression of PAI-1 in Mø, which is key to the protectiveeffect of CO in preventing lung ischemia reperfusion injury in mice.Exposure of SMC to serum did not alter expression of PAI-1 by westernblot (FIG. 12A). CO treatment slightly increased PAI-1 protein,suggesting that the anti-proliferative effect of CO does not involvedown-regulation of PAI-1. Similarly, PAI-1 protein expression inallogeneic aortas transplanted under CO treatment was similar to that ofallogeneic aortas transplanted in the absence of CO as tested byimmunohistology and Western blot (FIG. 12B). This suggests that theability of CO to modulate vascular injury associated with chronic graftrejection might not be directly linked to the expression of PAI-1.

CO is generated physiologically in most cell types through thecatabolism of heme by enzymes of the heme oxygenase family. Expressionof the inducible enzyme, HO-1, is a protective response to injury thatlimits the deleterious effects associated with inflammatory reactions.The protective effects of HO-1 are wide-ranging, including inhibition ofthe development of atherogenesis and intimal hyperplasia, and can inmany instances be mimicked by CO. The present studies demonstrate thatCO possesses direct vasoprotective properties. Continuous exposure tolow concentration of CO (250 ppm) suppresses development of intimalhyperplasia and graft infiltration by activated leukocytes (FIGS. 1A-1Iand 2A-2C) associated with transplant arteriosclerosis (FIG. 1A-1I).Exposure to CO (250 ppm) for just one hour prior to injury suppressesthe intimal hyperplasia associated with carotid artery angioplastyinjury in rats (FIGS. 3A-3G).

The physiologic relevance of the anti-proliferative effect of HO-1 inSMC, originally described in pulmonary epithelial cells, is supported bythe observation that SMC from HO-1 deficient mice proliferate morerapidly in vitro and in vivo than wild type SMC. The present datademonstrate that CO suppresses SMC proliferation in a manner similar toHO-1.

Generation of cGMP and the expression of p21^(Cip1) are essential forthe effects of CO, and are interrelated. The ability of CO toup-regulate p21^(Cip1) expression and suppress SMC proliferation isdependent on activation of guanylate cyclase (FIGS. 5A-5G). COup-regulates p21^(Cip1) (FIGS. 4A-4F) and the anti-proliferative effectof CO is dependent on the expression of p21^(Cip1) as this effect isabrogated in p21^(Cip1−/−) SMC (FIGS. 6A-6B). The ability of 8-Br-cGMPto suppress SMC proliferation was impaired in SMC derived fromp21^(Cip1) deficient mice (FIGS. 5A-5G), suggesting that cGMP suppressesSMC proliferation in vitro via the up-regulation of p21^(Cip1).

The individual contribution of cGMP and p38 MAPK to the effects of COseems to be cell type specific. The present data demonstrate that theanti-proliferative effect of CO in SMC is dependent on both signaltransduction pathways (FIGS. 5A-5G). CO requires cGMP for activation ofp38 MAPK, which is needed for CO to up-regulate p21^(Cip1) and suppressSMC proliferation. It is suggested that CO, like NO, activates p38 MAPKvia cGMP and activation of cGMP dependent protein kinases, consistentwith the observation that inhibition of PKGα and/or β suppresses theanti-proliferative effect of CO. The present data suggest that theanti-proliferative effect of CO is distinct from and can actindependently of NO. It should be noted that the link between cGMP, p38MAPK and p21^(Cip1) has not been reported in cells exposed to HO-1 orCO.

SMC express p21^(Cip1) upon vascular injury and recombinant adenovirusmediated p21^(Cip1) expression in SMC suppresses intimal hyperplasiafollowing vascular injury. The present data show that physiologicalexpression of p21^(Cip1) is involved in intimal hyperplasia: followingarterial injury, intimal hyperplasia in p21^(−/−) mice is three timeshigher as wild type controls (FIG. 4F), strongly supporting the notionthat CO induction of p21^(Cip1) expression in SMC (FIG. 5D) contributesto suppress intimal hyperplasia following vascular injury.

p21^(−/−) mice were used to assess the role of p21^(Cip1) expression onthe ability of CO to suppress intimal hyperplasia following arterialinjury, p21^(−/−) mice. One hour of CO pretreatment was sufficient tosuppress by more than 80% the development of intimal hyperplasia inp21^(−/−) mice or wild-type mice, as compared with their respectiveair-treated controls (FIG. 4F). One interpretation of these data is thatthe ability of CO to suppress neointimal proliferation in vivo actsindependently of p21^(Cip1) in SMC. In mice, in contrast with rats,there is a significant inflammatory/thrombotic process that presumablyleads to intimal proliferation. Given the potent anti-inflammatoryeffects of CO, it also seems possible that CO suppresses intimalhyperplasia in mice by inhibiting inflammation, a process that is almostcertainly independent of p21^(Cip1).

Given the known suppression of PAI-1 by CO in monocyte-macrophages andthe importance of PAI-1 in the pathogenesis of intimal hyperplasiafollowing vascular injury in some studies, whether a similar suppressionof PAI-1 occurred in the present system was investigated. No decrease inthe level of PAI-1 protein expression at 56 days after transplantationwas observed (FIG. 12B).

EXAMPLE 2 Protocols for the Treatment of Patients During Angioplasty andTransplantation Procedures

The following example illustrates protocols for treating patients duringangioplasty procedures and for treating a donor, organ, and/or recipientwith carbon monoxide during a transplantation procedure. Any one or moreof the following procedures may be used in a given transplantationprocedure.

Angioplasty

CO can be administered systemically or locally to a patient prior to,during, and/or after an angioplasty procedure is performed in thepatient. Treatment can be administered at doses varying from 10 ppm to1000 ppm (e.g., about 100 ppm to about 800 ppm, about 150 ppm to about600 ppm (e.g., about 150 ppm), or about 200 ppm to about 500 ppm (e.g.,about 250 ppm or about 500 ppm)). For example, CO can be administered tothe patient, intermittently or continuously, starting 0 to 20 daysbefore the procedure is performed, e.g., starting at least about 30minutes, e.g., about 1, 2, 3, 5, 7, or 10 hours, or about 1, 2, 4, 6, 8,10, 12, 14, 18, or 20 days, or greater than 20 days, before theprocedure. Alternatively or in addition, CO can be administered to thepatient during the procedure, e.g., through an instrument used toperform the angioplasty and/or by inhalation. Alternatively or inaddition, CO can be administered to the patient after the procedure,e.g., starting immediately after completion of the procedure, andcontinuing for about 1, 2, 3, 5, 7, or 10 hours, or about 1, 2, 5, 8,10, 20, 30, 50, or 60 days, or indefinitely, after the completion of theprocedure.

Transplantation

Treatment of a Donor

Prior to harvesting an organ or tissue, the donor can be treated withinhaled carbon monoxide (250 ppm) for one hour. Treatment can beadministered at doses varying from 10 ppm to 1000 ppm for times varyingfrom one hour to six hours, or for the entire period from the momentwhen it becomes possible to treat a brain-dead (cadaver) donor to thetime the organ is removed. Treatment should start as soon as possiblefollowing the declaration that brain death is present. In someapplications, it may be desirable to begin treatment before brain death.

For non-human animals (e.g., pigs) to be used as xenotransplantationdonors, the live animal can be treated with relatively high levels ofinhaled carbon monoxide, as desired, so long as the carboxyhemoglobin soproduced does not compromise the viability and function of the organ tobe transplanted. For example, one could use levels greater than 500 ppm(e.g., 1000 ppm or higher, and up to 10,000 ppm, particularly for brieftimes).

Treatment of the Organ In Situ

Before an organ is harvested from a donor, it can be flushed with asolution, e.g., a buffer or medium, without red blood cells while it isstill in the donor. The intent is to flush the organ with a solutionsaturated with carbon monoxide and maintained in a carbon monoxideatmosphere so that the carbon monoxide content remains at saturation.Flushing can take place for a time period of at least 10 minutes, e.g.,1 hour, several hours, or longer. The solution should ideally deliverthe highest concentration of carbon monoxide possible to the vasculatureof the organ.

Treatment of an Organ or Tissue

The organ or tissue can be preserved in a medium that includes carbonmonoxide from 20 the time it is removed from the donor to the time it istransplanted to the recipient. This can be performed by maintaining theorgan or tissue in the medium comprising CO, or by perfusing it withsuch a medium. Since this occurs ex vivo rather than in an animal, veryhigh concentrations of CO gas can be used (e.g., 10,000 ppm) to keep themedium saturated with CO.

Treatment of a Recipient

The recipient can be treated with carbon monoxide. Treatment can beginon any day before the transplantation procedure, e.g., on the day of thetransplantation procedure at least one hour before surgery begins.Alternatively, it could begin at least 30 minutes before re-perfusion ofthe organ in the recipient. It can be continued for at least 30 minutes,e.g., 1 hour. Carbon monoxide doses between 10 ppm and 3000 ppm can bedelivered for varying times, e.g., minutes or hours, and can beadministered on the day of and on days following transplantation. Forexample, a recipient can inhale a concentration of carbon monoxide,e.g., 3000 ppm, for three consecutive 10 second breath holds.Alternatively, the recipient can inhale, say 200 ppm for an extendedtime, such as 20 days. Carboxyhemoglobin concentrations can be utilizedas a guide for appropriate administration of carbon monoxide to apatient. Usually, treatments for recipients should not raisecarboxyhemoglobin levels above those considered to pose an acceptablerisk for a patient in need of a transplant.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-43. (canceled)
 44. A kit comprising: (a) an angioplasty device; and(b) a vessel containing a carbon monoxide composition.
 45. The kit ofclaim 44, wherein the angioplasty device is capable of administeringcarbon monoxide to a patient.
 46. The kit of claim 44, furthercomprising: (c) instructions for use of the carbon monoxide compositionin a method for performing angioplasty in a patient.
 47. The kit ofclaim 44, wherein the carbon monoxide composition is a liquidcomposition.
 48. The kit of claim 44, wherein the carbon monoxidecomposition is a gaseous composition.
 49. An angioplasty devicecomprising: an inflatable member comprising a plurality of apertures;and a reservoir comprising carbon monoxide gas and connected to theinflatable member, whereby the gas is provided to the inflatable memberduring inflation of the inflatable member.